saspase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing

SASPase regulates stratum corneumhydration through profilaggrin-to-filaggrinprocessing

Takeshi Matsui1*y, Kenichi Miyamoto2, Akiharu Kubo3,4, Hiroshi Kawasaki3, Tamotsu Ebihara3,Kazuya Hata5, Shinya Tanahashi5, Shizuko Ichinose6, Issei Imoto7,8, Johji Inazawa7,Jun Kudoh2, Masayuki Amagai3

Keywords: dermatology; epidermis;

filaggrin; protease; skin

DOI 10.1002/emmm.201100140

Received September 22, 2010

Revised March 11, 2011

Accepted March 11, 2011

The stratum corneum (SC), the outermost layer of the epidermis, acts as a barrier

against the external environment. It is hydrated by endogenous humectants to

avoid desiccation. However, the molecular mechanisms of SC hydration remain

unclear. We report that skin-specific retroviral-like aspartic protease (SASPase)

deficiency in hairless mice resulted in dry skin and a thicker and less hydrated SC

with an accumulation of aberrantly processed profilaggrin, a marked decrease of

filaggrin, but no alteration in free amino acid composition, comparedwith control

hairless mice. We demonstrated that recombinant SASPase directly cleaved a

linker peptide of recombinant profilaggrin. Furthermore, missense mutations

were detected in 5 of 196 atopic dermatitis (AD) patients and 2 of 28 normal

individuals. Among these, the V243A mutation induced complete absence of

protease activity in vitro, while the V187I mutation induced a marked decrease in

its activity. These findings indicate that SASPase activity is indispensable for

processing profilaggrin and maintaining the texture and hydration of the SC. This

provides a novel approach for elucidating the complex pathophysiology of atopic

dry skin.

INTRODUCTION

Skin is the outermost tissue of terrestrial animals. It forms an

effective barrier between the organism and the environment,

which is indispensable for the prevention of the invasion of

microorganisms, chemical compounds and allergens, and the

maintenance of moisture levels of the skin. Skin is composed of

three layers: the epidermis, dermis, and hypodermis. The

epidermis is a stratified squamous, keratinized epithelium

composed of the stratum basale (SB), stratum spinosum (SS),

stratum granulosum (SG), and stratum corneum (SC) (Watt,

1989). Epidermal cells (keratinocytes) divide and differentiate

as they move upward from the SB to the SC. At the SG-to-SC

transition, the keratinocytes dramatically transform themselves

from three-dimensional living cells to two-dimensional, flat-

tened and dead cells without intracellular organelles containing

keratin bundles and lipids to constitute the SC (Candi et al,

2005). During this abrupt and dynamic terminal differentiation,

keratinocytes express various proteins such as keratins,

profilaggrin/filaggrin, involucrin, small proline-rich proteins,

Research ArticleSASPase regulates stratum corneum hydration

(1) Medical Top Track (MTT) Program, Medical Research Institute, Tokyo

Medical and Dental University, Tokyo, Japan

(2) Laboratory of Gene Medicine, Keio University School of Medicine, Tokyo,

Japan

(3) Department of Dermatology, Keio University School of Medicine, Tokyo,

Japan

(4) Center for Integrated Medical Research, Keio University School of

Medicine, Tokyo, Japan

(5) Sunplanet Co. Ltd, Gifu, Japan

(6) Instrumental Analysis Research Center, Tokyo Medical and Dental

University, Tokyo, Japan

(7) Department of Molecular Cytogenetics, Medical Research Institute and

School of Biomedical Science, Tokyo Medical and Dental University,

Tokyo, Japan

(8) Department of Human Genetics and Public Health, Institute of Health

Biosciences, The University of Tokushima Graduate School, Tokushima,

Japan

*Corresponding author: Tel: þ81 75 753 9844; Fax: þ81 75 753 9820;

E-mail: [email protected] Present address: Institute for Integrated Cell – Material Sciences (iCeMS),

Kyoto University, Kyoto, Japan

320 � 2011 EMBO Molecular Medicine EMBO Mol Med 3, 320–333 www.embomolmed.org

loricrin, cystatin A, and elafin, which form the cornified

envelope of mature corneocytes (Candi et al, 2005).

It has long been recognized that there is a heritable

component for the development of atopic dermatitis (AD)

(Barnes, 2010). Recent reports indicate that ‘filaggrin’ has

nonsense mutations in ichthyosis vulgaris (IV) patients and is

the major predisposing factor for atopic eczema, asthma, and

allergies (Smith et al, 2006; Irvine, 2007; Palmer et al, 2006;

Sandilands et al, 2009). These reports further indicate that early

onset of AD is caused by outside-to-inside paradigms, namely a

primary barrier abnormality (Elias & Steinhoff, 2008), and

filaggrin nonsense mutations are carried by nearly 10% of

Europeans. However, such filaggrin mutations are found in�50

and 20% of European and Japanese AD patients, respectively

(Barker et al, 2007; Nomura et al, 2008; Sandilands et al, 2007,

2009), indicating that patients with AD who have normal

filaggrin alleles are likely affected by other previously

unidentified predisposing factors.

Filaggrin is expressed as a profilaggrin of >400 kDa in

humans, which is a major component of keratohyalin granules

in the SG of the epidermis (Dale et al, 1985; Presland et al, 2006).

Profilaggrin is an insoluble phosphoprotein that consists of an

amino terminal Ca2þ-binding protein of the S-100 family, linked

to 10–12 tandem filaggrin monomer repeats (Dale et al, 1985;

Harding & Scott, 1983; McGrath & Uitto, 2008). At the SG-to-SC

transition, each filaggrin repeat is processed by certain

protease(s) to generate the filaggrin monomer (37 and 28 kDa

in human and mouse, respectively). The resulting monomer

filaggrin has an unusual cationic charge, strongly binds to and

bundles the keratin cytoskeleton to form microfibrils, and is

believed to contribute to the production of flattened cells in the

lower SC (Dale et al, 1978). Additionally, part of the filaggrin–

keratin complex is cross-linked by transglutaminase to build the

skin barrier (Candi et al, 1998, 2005; Harding & Scott, 1983;

Steinert & Marekov, 1995). Keratin-bound filaggrin is citrulli-

nated and further degraded into amino acids, which constitute a

part of the natural moisturizing factor (NMF) in the upper SC

(Tarcsa et al, 1996; Mechin et al, 2005; Nachat et al, 2005;

Ishida-Yamamoto et al, 2002; Denecker et al, 2007; Kamata et al,

2009). Therefore, at the transition of the SG-to-SC, processing

from profilaggrin to filaggrin is the rate-limiting, critical step for

the profilaggrin processing cascade leading to SC moisturizing.

Several proteases are involved in profilaggrin to filaggrin

processing. Calpain I and profilaggrin endopeptidase I (PEP-I)

are important for the processing of the linker peptides between

the filaggrin monomer repeats to generate the monomeric

filaggrin in vitro (Resing et al, 1989, 1993a,b, 1995; Yamazaki

et al, 1997); furin or convertase are involved in cleavage of

the N-terminus from profilaggrin (Pearton et al, 2001); and

knockout mice of matriptase/MT-SP1 and prostasin (CAP1/

Prss8) show a defect in the conversion of profilaggrin to filaggrin

(Leyvraz et al, 2005; List et al, 2003). Although the N- and

C-terminal sequences of mouse, rat and human filaggrin have

been determined (Resing et al, 1989, 1993b; Thulin & Walsh,

1995; Thulin et al, 1996), whether the linker sequences of

profilaggrin are cleaved by matriptase or prostasin remains

unclear.

Human and mouse retroviral-like aspartic protease, SASPase

(skin aspartic protease; Asprv1), was the first identified

stratified epithelia-specific protease expressed exclusively in

the SG (Bernard et al, 2005; Matsui et al, 2006). This protease

has been cloned as a 12-O-tetradecanoylphorbol-13-acetate

(TPA)-induced gene (Taps) from mouse back skin epidermis

(Rhiemeier et al, 2006). Recently, aberrant SASPase expression

in transgenic mice was reported to cause impaired skin

regeneration and skin remodelling after cutaneous injury and

chemically induced hyperplasia (Hildenbrand et al, 2010). We

have previously reported that SASPase-deficient mice showed

increased fine wrinkles on the side of the adult body, suggesting

that SASPase plays a certain physiological role in the SG and SC,

although precise physiological and biochemical analysis was

difficult due to the presence of hair. In this report, we produced

SASPase-deficient ‘hairless’ mice. Through physiological and

biochemical analyses of the mice, we demonstrate that SASPase

activity plays a key role in determining the texture of SC by

modulating SC hydration as well as profilaggrin processing.

Moreover, we also report the identification of loss-of-function

mutation of SASPase in the human genome.

RESULTS

Generation of SASPase-deficient hairless mice

Using high-throughput in situ hybridization screening, we

previously identified a mouse homologue of SASPase and

showed that SASPase-deficient mice displayed fine wrinkles on

the side of their bodies (Matsui et al, 2004, 2006). Because

SASPase is expressed exclusively in the SG and SC, these

wrinkles were hypothesized to be derived from aberrant

functions of the SG and SC. To further analyse the effect of a

deficiency of SASPase on the epidermal surface, we transferred

the ablated allele to a Hos/HR-1 hairless background. Hos:HR-1

mice (BALB/c background) were crossed with SASP�/� mice

(C57BL/6J background) using a speed congenic method

(Wakeland et al, 1997). Immunoblotting of the skin in

SASPþ/þ, SASPþ/�, and SASP�/� hairless mice showed no

expression of SASPase in the SASP�/� epidermis (Supporting

information Fig 1A). Frozen sections of SASPþ/� and SASP�/�

hairless mouse ears were stained with anti-SASP pAb and

revealed a loss of the SASPase signal in the SG of the SASP�/�

epidermis (Supporting information Fig 1B). Therefore, immu-

noblotting and immunofluorescence staining of frozen sections

did not detect SASPase in SASPase deficient mice, indicating that

the epidermis of SASP�/� mice was successively deficient in

SASPase protein in the hairless background.

Appearance of SASPS/S hairless mice

Like normal mice, hairless mice (Hos:HR-1) grow hair after

birth; however, at 17–23 days, they lose this hair due to hair root

atrophy in 3–4 days, starting with the head, and become

completely hairless at 3.5 weeks of age. Thereafter, we observed

that SASP�/� hairless mice had more fine wrinkles and drier,

rougher skin than their SASPþ/þ and SASPþ/� counterparts

(Fig 1A left). Female mice tended to show a more marked

Research ArticleTakeshi Matsui et al.

www.embomolmed.org EMBO Mol Med 3, 320–333 � 2011 EMBO Molecular Medicine 321

phenotype than male mice (data not shown). Stereomicroscopic

examination revealed that SASPþ/þ and SASPþ/� mice had

smooth, moist-looking skin with fine grooves, whereas SASP�/�

hairless mice had skin with raised scales composed of many

horny cells (Fig 1A right). When hung by their tails, more

apparent differences were observed (Fig 1B). Specimens were

fixed by standard techniques, dehydrated, dried at a critical

point and examined employing scanning electron microcopy

(SEM). After drying, epithelial scales of the skin of the SASPþ/�

hairless mice had fallen off, exposing the surface structure and

resulting in many fine grooves. In contrast, coarse grooves were

observed in the SASP�/� hairless mice (Fig 1C). Similarly, SEM

revealed that the individual horny cells were easily identified in

SASPþ/� hairless mice, whereas the intercellular spaces

between the horny cells could not be readily discerned in their

SASP�/� counterparts (Fig 1D).


Figure 1. Morphological characterization of the

epidermis of SASPase-deficient hairless mice.

A. Macroscopic observation of SASPþ/þ, SASPþ/� and

SASP�/� hairless mice (left). Note that SASP�/� hairless

mice showed drier and rougher skin. Stereomicro-

scopic examination of SASPþ/� and SASP�/� hairless

mice epidermis are also shown (right). Note that

SASP�/� hairless mice had an epidermis with raised

scales composed of many horny cells, whereas SASPþ/�

hairless mice had smooth, moist-looking skin with fine

grooves.

B. Fine wrinkle formation in hung SASP�/� hairless mice.

When hung by the tail, more wrinkles appeared on the

surface of the back skin in SASP�/� hairless mice

compared with SASPþ/� hairless mice.

C. Stereomicroscopic examination of critical point dried

epidermis of SASPþ/� and SASP�/� hairless mice. In the

SASPþ/� hairless mice epidermis, many fine grooves

were observed, whereas in the SASP�/� hairless mice,

coarse grooves were observed. White boxes indicate

the areas of interest investigated by SEM in D.

D. SEM of the epidermis of SASPþ/� and SASP�/� hairless

mice. In SASP�/� hairless mice, the intercellular spaces

between horny cells could not be readily discerned,

whereas individual horny cells were identified in their

SASPþ/� counterparts. Scale bars: 333mm (left) and

375mm (right).


Morphology of SASPS/S mice epidermis

Next, we examined the histological analysis of SASP�/� hairless

mice. The epidermis of the SASP�/� mice showed a normal layer

organization of keratinocytes, and keratohyalin-granules were

observed in both SASPþ/þ and SASP�/� mice at the level of

hematoxylin–eosin (HE)-stained paraffin section images (Fig 2A

and B). However, the cornified cells of the SASP�/� SC were

compacted more tightly, and the number of layers was increased

compared with SASP þ/þ mice (Fig 2A and B). Ultrathin section

electron microscopy confirmed these findings at a higher resolu-

tion (Fig 2C–G). The appearance of individual cells from the SB to

SS was indistinguishable between the SASPþ/þ and SASP�/� skin,

and the SG contained both F- and L-granules in the SASP�/� SC

(Fig 2C andD). Furthermore, the SC of SASP�/�mice showed 7–10

layers of electron dense and tightly compacted cell layers com-

pared with that of SASPþ/þ, which usually exhibited 5–7 layers

(Fig 2C–H). These results suggest that a deficiency of SASPase in

hairless background mice induced an abnormality in the SC.

Physiological features of SASPS/S epidermis

To physiologically characterize the epidermal surface of the

SASP�/� hairless mice, we measured the trans-epidermal water

loss (TEWL) and the SC hydration of SASPþ/þ (n¼ 7) and

SASP�/� (n¼ 11) mice (Fig 3A and B). Although the TEWL was

not significantly changed between SASPþ/þ and SASP�/� mice

(Fig 3A), a clear difference was observed in SC hydration. As

such, SC hydration of SASP�/� mice was significantly lower

than in SASPþ/þ mice (Fig 3B). These results indicate that

SASP�/� hairless mice showed markedly decreased SC hydra-

tion without alteration of barrier function.

Aberrant profilaggrin processing in SASPS/S hairless mice

Next, to analyse the epidermal differentiation of SASP�/�

hairless mice, the expression of various epidermal differentia-

tion markers were examined. Immunofluorescence staining of

frozen sections of back skin epidermis with anti-keratin 14,

keratin 1, involucrin and loricrin pAbs revealed normal


Figure 2. SASPS/S hairless mouse epidermis

showed an increased number of layers in the

aberrant SC.

A, B. Sections of SASPþ/þand SASP�/�epidermis

examined by HE staining. HE staining of

SASP�/� hairless mice showed a marked

thickening of the SC with an increased

number of layers compared with SASPþ/þ

mice. Scale bar: 100mm.

C, D. Sections of SASPþ/þ and SASP�/�epidermis

examined by electron microscopy. TEM

analysis of SASPþ/þ and SASP�/� epidermis

showed an increased number of electron

dense layers in the SC of SASP�/� mice

compared with SASPþ/þ mice. Scale bar:

4mm.

E. Typical layers of the SC of SASPþ/þ hairless

epidermis. Scale bar: 500 nm.

F. The granular layer of the SASP�/� hairless

mice. Both F-granules (F) and L-granules (L)

were observed, suggesting that keratohyalin

granule formation normally occurs in

SASP�/� hairless mice. Scale bar: 2mm.

G. Typical electron dense layers of the SC of

SASP�/� hairless epidermis. Scale bar:

100 nm.

H. The number of layers in the SC of SASPþ/þ and

SASP�/� epidermis were counted in five

independent areas/mouse in the TEM images

of two female mice. The average number of

layers in the SC was 5.8� 0.8 SD and

8.6�1.07 SD in SASPþ/þ and SASP�/�

epidermis, respectively.


expressions and localization of epidermal differentiation

markers (Supporting information Fig 2A). Immunoblotting of

back skin epidermal urea extracts with anti-keratin 14, keratin 1,

involucrin and loricrin pAbs also revealed that the correspond-

ing expression levels were not altered (Supporting information

Fig 2B). On the other hand, immunofluorescence staining

with anti-filaggrin pAb revealed that filaggrin-positive layers

of the SC (lower SC) were increased in the back skin epidermis

of the SASP�/� hairless mice (Fig 4A and B). To carefully

compare the filaggrin in the lower SC, we tape-stripped the

SC of the SASPþ/� and SASP�/� epidermis. Coomassie brilliant

blue (CBB) staining of equivalent amounts of the extracts

revealed that all the major bands were decreased, suggesting

there were increased concentrations of certain smear

proteins (Fig 4C, left). Immunoblotting of the same samples

with anti-filaggrin pAb revealed the accumulation of primarily

two smear bands below the size of dimeric and trimeric filaggrin

and that a mature filaggrin band was rarely detected (Fig 4C,

right). These results suggest that a deficiency of SASPase

resulted in the accumulation of premature processed dimeric

and trimeric filaggrin and that mouse SASPase cleaves the

linker sequence of mouse profilaggrin in vivo. The processing

of mouse profilaggrin in the C57BL/6J mouse was reported to

occur in a two-step process via two types of profilaggrin

linker sequences with or without FYPV, respectively (Resing

et al, 1989). First, a profilaggrin linker sequence containing

FYPV may be cleaved, resulting in the accumulation of a two-

domain intermediate (2DI) and a three-domain intermediate

(3DI). Second, the linker type without FYPV, which connects

2DI and 3DI of monomeric filaggrin, is potentially cleaved by a

Ca2þ dependent protease (Resing et al, 1989, 1993a). Some

amino acid residues are then removed from the exposed sites by

further exoprotease activity (Resing et al, 1989). Therefore,

accumulation of dimeric and trimeric-like profilaggrin in the

SASP�/� epidermis in Hos:HR-1 background suggests that

SASPase may be involved in either the first or second processing

steps.

SASPase directly cleaves the profilaggrin linker sequence

in vitro

Accumulation of aberrant dimeric and trimeric profilaggrin in

the lower SC of the SASP�/� hairless mice implies that the

profilaggrin linker peptide is a direct substrate for SASPase. To

confirm this hypothesis, we examined whether SASPase directly

cleaves the profilaggrin linker peptide in vitro. Because it was

difficult to produce recombinant mouse filaggrin with a linker

peptide in Escherichia coli because of excess degradation, we

produced human filaggrin (hFilaggrin) as a fusion protein of

maltose binding protein (MBP) connected with the human

profilaggrin linker peptide in E. coli. Purified MBP-hFilaggrin

showed a triple banding pattern. The N-terminal amino acid

sequence of the major upper two bands revealed that both

proteins had an intact N-terminus of MBP indicating that

C-terminally processed filaggrin (MBP-hFilaggrin-DC) was

copurified with MBP-hFilaggrin. To prepare the active form

of SASPase (14 kDa; hSASP14; 191-326aa), we expressed and

purified a 28 kDa form of human SASPase (hSASP28; 85-343aa)

as a fusion protein with GST (GST-hSASP28) in E. coli (Bernard

et al, 2005). After complete autoprocessing under weakly acidic

conditions (pH 6.0), which is the optimum pH of SASPase, the

produced hSASP14 was purified (Fig 5A; Bernard et al, 2005;

Matsui et al, 2006). Next, hSASP14 was incubated with MBP-

hFilaggrin/-hFilaggrin-DC under weakly acidic conditions

(pH 6.0). As shown in Fig 5B, MBP-hFilaggrin/hFilaggrin-DC

was cleaved into mainly 42, 37 and 23 kDa proteins (Fig 5B

and C). The 42 kDa protein was recognized by anti-MBP pAb

(data not shown), and the N-terminal amino acid sequencing

confirmed that the protein at 42 kDa was the MBP protein itself.

The N-terminal amino acid sequencing of the 37 and 23 kDa

identified the same peptide, ‘QVSTH’. This N-terminal amino


Figure 3. SASPase regulates SC hydration.

A. Trans-epidermal water loss (TEWL) of SASPþ/þ

and SASP�/� hairless mice. TEWL of hairless mice

were measured using VAVO SCAN (Asahi Biomed,

Tokyo, Japan). The TEWL of SASP�/� hairless mice

was not significantly changed. The numbers of

animals tested were: SASPþ/þ, n¼7 and SASP�/�,n¼ 11. ns: not significant (Mann Whitney test on

mean values� SD using Graphpad software).

B. SC hydration levels of SASPþ/þ and SASP�/�

hairless mice. SC hydration levels of hairless mice

were measured using ASA-M1 (Asahi Biomed).

The SC hydration of SASP�/� hairless mice was

significantly lower than that of SASPþ/þ hairless

mice. The numbers of animals tested were:

SASPþ/þ, n¼ 7 and SASP�/�, n¼ 11. The p value is

indicated above the bar (Mann Whitney test on

mean values� SD using Graphpad software).


acid sequence corresponded to the previously reported

N-terminal amino acid sequence of native monomeric human

filaggrin, in which Q ismodified into pyrrolidone carboxylic acid

(PCA) (Thulin & Walsh, 1995; Thulin et al, 1996). These results

indicate that hSASP14 cleaved MBP-hFilaggrin and produced

MBP (42 kDa) and hFilaggrin (37 kDa), of which the N-terminus

was QVSTH. Furthermore, MBP-hFilaggrin-DC was cleaved at

the same site and produced MBP (42 kDa) and hFilaggrin-DC

(23 kDa), of which the N-terminus was QVSTH (Fig 5B and C).

Thus, we conclude that hSASP14 cleaved the linker sequence


Figure 4. Aberrant expression of filaggrin in SASPS/S hairless mice.

A. Immunofluorescence staining of frozen sections of the back skin of SASPþ/� and SASP�/� mice stained with anti-filaggrin antibody (red). Nuclei were

counterstained with bisbenzimide (blue). The SASP�/� epidermis showed an increased amount of filaggrin-positive stained lower SC. Dashed lines represent

the border between the epidermis and dermis. BF, bright field. Scale bar: 10mm.

B. Enlarged view of A shows an increased amount of lower SC layers in the SASP�/� hairless epidermis.

C. Equivalent amounts of tape-stripped extracts (10 times, 5mg) from SASPþ/þ (n¼2), SASPþ/� (n¼2), and SASP�/� (n¼ 2) mice were immunoblotted with

anti-filaggrin antibodies. CBB staining of extracts revealed a reduced expression of filaggrin monomer bands and other major SC proteins in the SASP�/�

hairless mice epidermis (left; Coomassie). Immunoblotting with anti-filaggrin demonstrated that an accumulation of aberrant filaggrin degradation products

(dimer and trimer sized) was detected (right), whereas mature filaggrin was rarely detected. As equivalent amounts of SC extracts were loaded, the intensity of

the profilaggrin band was decreased in SASP�/�, possibly due to an increase in the concentrations of other smear proteins.


of human profilaggrin between ‘GSFLY’ and ‘QVSTH’ in vitro

(Fig 5C and D).

Normal free amino acid composition in SC of SASPS/S mice

Previous reports suggest that NMFs serve as natural humectants

of SC (reviewed in Rawlings & Matts, 2005). To investigate

whether aberrant processing of filaggrin had a detrimental effect

on the composition of NMFs, we performed free amino acid

analysis of tape stripped SC of SASPþ/þ (n¼ 7) and SASP�/�

(n¼ 11) mice. There were no alterations in the total amount or

the composition of free amino acids (Fig 6A and B). These

results suggest the NMFs were normal in SASP�/� SC, despite

the marked decrease in SC hydration.

Mutations of human SASPase affect autoprocessing and

profilaggrin linker cleavage activity

Analysis of our SASP�/� hairless mice indicates that loss of

SASPase activity may result in dry skin accompanied by an

accumulation of unprocessed profilaggrin. Thus, we investi-

gated whether the human genome possesses SASPase

mutations, which consequently affect protease activity, i.e.

profilaggrin to filaggrin processing activity. We investigated

mutations in the SASPase gene in 28 control subjects and 196

AD-patients (Sasaki et al, 2008). As a result of the mutation

search on the human SASPase gene, two types of missense

mutations (D232Y and V243A: 3.5% [1/28]) in the control

subjects, four types of missensemutations (A54S: 0.5% [1/196],


Figure 5. Recombinant hSASP14 directly cleaves recombinant filaggrin in vitro.

A. Production and purification of hSASP14 by autoprocessing of GST-hSASP28. Purified GST-hSASP28 (arrow) was incubated for the indicated times (00 , 0min; 600 ,60min) with 700mM NaCl at pH 6.0. GST-hSASP28 underwent autoprocessing and produced hSASP14 (arrowhead). Cleaved GST-fusion proteins were

removed by passing through Glutathione Sepharose 4B beads to purify hSASP14 (arrowhead). Asterisk indicates a dimer of hSASP14.

B. Cleavage of profilaggrin linker peptide by hSASP14 in vitro. The purified MBP-hFilaggrin/MBP-hFilaggrin-DC (arrow) was incubated with or without the

purified hSASP14 (arrowhead) with 700mM NaCl at pH 6.0 for 60min at 378C. The linker peptide of profilaggrin between MBP and hFilaggrin in

MBP-hFilaggrin/MBP-hFilaggrin-DC was cleaved by hSASP14, resulting in the production of MBP (42 kDa), hFilaggrin (37 kDa), and hFilaggrin-DC (23 kDa).

The N-terminal amino acid sequencing of hFilaggrin (37 kDa) and hFilaggrin-DC (23 kDa) protein identified QVSTH amino acids, which corresponded to the

linker peptide of profilaggrin.

C. Schematic representation of the mode of processing of MBP-hFilaggrin by hSASP14 as described in B.

D. Schematic representation of the possible cleavage site of profilaggrin by hSASP14. Homodimerized hSASP14 proteins were suggested to primarily cleave

between GSFLY-QVSTH in the profilaggrin linker sequence (arrow).


I186T: 0.5% [1/196], V187I: 1.5% [3/196], R311C: 0.5% [1/

196]), and three types of silent mutations (F101F: 0.5% [1/196],

P206P: 3.1% [6/196], N276N: 1.5% [3/196]) in the AD patients

were identified (Fig 7A, Supporting information Tables I and II).

All mutations were heterozygous. V187I was most frequently

identified (identified in three AD patients). A54S and R311C

were found in the same patient and in the same allele. The

number of patients and mutation types are shown in Supporting

information Tables I and II.

To examine whether these mutations affect the protease

activity of SASPase, we bacterially expressed and purified

GST-hSASP28 bearing the mutations I186T, V187I, R311C,

D232Y, or V243A, and performed an in vitro autoprocessing

assay as previously described (Bernard et al, 2005; Matsui et al,

2006). To compare the autoprocessing activity in vitro, we

purified GST-hSASP28 mutants (Fig 7B). We could not examine

the autoprocessing activity of GST-hSASP28(I186T)/(R311C),

because full-length GST-hSASP28(I186T)/(R311C) was barely

purified, possibly due to the higher autoprocessing activity in

E. coli. The rest of the purified GST-hSASP28(WT)/(D232Y)/

(V243A) was incubated under weakly acidic conditions

(pH 6.0), which is the optimal pH of SASPase (Bernard

et al, 2005; Matsui et al, 2006). As shown in Fig 7C,

GST-hSASP28(D232Y) showed autoprocessing activity similar

to that of the WT, and V243A showed no autoprocessing

activity.

We next examined whether the V187I mutation, which was

identified most frequently in AD patients (three patients),

affected autoprocessing activity in vitro. The purified GST-

hSASP28(WT)/(V187I) was incubated under weakly acidic

conditions (pH 6.0). As shown in Fig 7D, GST-hSASP28(V187I)

showed decreased autoprocessing activity in both 300 and

400mM NaCl. Fig 7E shows the time course by semi-

quantification of produced hSASP14. Because V187I is located

outside of the cleaved hSASP14, autoprocessed hSASP14 from

hSASP28(V187I) should be the same as the WT. Therefore, the

initial autoprocessing reaction (1–30min in 300mM NaCl)

should reflect the difference between WT and V187I. The

reaction rate of GST-hSASP28(V187I) at 300mM NaCl was

estimated at 5.6-fold over 15–30min, which is lower than that of

GST-hSASP28(WT). These results indicate that hSASP28(V187I)

has substantially reduced activity at pH6.0 in vitro compared to

WT. Finally, we examined whether the V187I mutation had

an effect on the filaggrin linker cleavage activity of GST-

hSASP28 in vitro. To examine whether the mutation of V187I

affected the profilaggrin cleavage activity, we incubated GST-

hSASP28(WT) or GST-hSASP28(V187I) with MBP-hFilaggrin/

MBP-hFilaggrin-DC under weakly acidic conditions (pH 6.0).

As expected, the MBP-hFilaggrin cleavage activity by GST-

hSASP28 was suppressed in GST-hSASP28(V187I) compared

with GST-hSASP28(WT), relative to the production of hSASP14

(Fig 7E). These results indicate that GST-hSASP28(V187I)

showed decreased autoprocessing activity at pH 6.0, resulting

in decreased profilaggrin linker cleavage activity in vitro.

It is important to elucidate whether these heterogenic loss-of-

function mutations of SASPase affect human skin physiology.

And it is possible there was some dry skin in our non-AD cohort

as criteria of this cohort did not discriminate against dry skin

(Sasaki et al, 2008). We measured TEWL and SC hydration of a

non-AD individual with a mutation (V243A/þ) as well as three

normal individuals without mutations. The appearance of the

skin surface, TEWL and SC hydration of V243A/þ were similar

to the three normal individuals (Supporting information Fig 3).

These results did not provide conclusive evidence due to the

small numbers tested. In the case of complex disorders like AD,

it is difficult to prove a direct role of the sequence variant in the


Figure 6. Free amino acids in SC of SASPR/R and SASPS/S mice.

A. Total free amino acid composition analysis. Tape stripped SC of SASPþ/þ (n¼7, filled column) and SASP�/� (n¼11, open column) hairless mice were subjected

to amino acid analysis. Total free amino acid composition analyses showed no difference between the SC of SASPþ/þ and SASP�/� mice. ns: not significant

(Mann–Whitney test on mean values� SD using Graphpad software).

B. Individual free amino acid composition analysis. Individual free amino acid composition analyses also showed no difference between the SC of SASPþ/þ(n¼7,

filled columns) and SASP�/� mice (n¼ 11, open columns). ns: not significant (Mann–Whitney test on mean values� SD using Graphpad software).



Figure 7. Biochemical characterization of human mutations in SASPase.

A. Schematic representation of human mutations identified in AD patients (n¼196; closed circles) and case controls (n¼ 28; open circles). The amino acid

sequence of the autoprocessing site (between Asn190 and Ser191) is indicated. Asterisks of A54S and R311C indicate that these mutations were found in the

same patient and in the same allele.

B. Purification of GST-hSASP28 mutants. Purified GST-hSASP28 (WT)/(I186T)/(V187I)/(R311C)/(D232Y)/(V243A) (1mg) were subjected to SDS-PAGE and stained

with CBB. GST-hSASP28 (V187I) and (V243A) did not produce any partially cleaved products. Arrow indicates GST-hSASP28. Asterisk indicates the partially

cleaved product of N-terminal hSASP28 (85-190aa) fused with GST (see A).

C. Comparison of autoprocessing activity among GST-hSASP28(WT), (D232Y) and (V243A). 1.4mg/ml of GST-hSASP28(WT), (D232Y) and (V243A) were incubated

at pH 6.0 for indicated times and subjected to SDS-PAGE and stained with CBB. The autoprocessing activity of GST-hSASP28(D232Y) was comparable to that of

GST-hSASP28(WT), whereas GST-hSASP28(V243A) did not show any autoprocessing activity in vitro. Arrowhead indicates the hSASP14.

D. Comparison of autoprocessing activity between GST-hSASP28(WT) and (V187I). 0.9mg/ml of GST-hSASP28(WT) and (V187I) were incubated under 300 or

400mMNaCl at pH 6.0 for indicated times, subjected to SDS-PAGE, and stainedwith CBB. The autoprocessing activity of GST-hSASP28(V187I) was weaker than

that of GST-hSASP28(WT) in both conditions.

E. Reaction curve of GST-hSASP28(WT) and (V187I) autoprocessing. Autoprocessed hSASP14 from GST-hSASP28(WT) (closed circles) and GST-hSASP28(V187I)

(open circles) in Fig 8D was semi-quantified from a gel image. The reaction rate was higher in 400mM (right) than in 300mM NaCl (left). V187I mutation

affected the initial reaction rate and showed 5.6-fold reduced activity over 15–30min in 300mM NaCl.

F. Comparison of profilaggrin linker peptide cleavage activity between GST-hSASP28(WT) and (V187I). 0.9mg/ml of GST-hSASP28(WT) and (V187I) were

incubated with 0.4mg/ml of MBP-hFilaggrin/MBP-hFilaggrin-DC under 300 or 400mM NaCl at pH 6.0 for the indicated times, subjected to SDS-PAGE, and

stained with CBB. The profilaggrin linker cleavage activity was weaker in GST-hSASP28(V187I) than in that of (WT), relative to the production of hSASP14 by

autoprocessing. An enhanced image of the MBP and hFilaggrin band areas is also shown.


pathogenesis. In the future, it is necessary to perform a

large-scale cohort analysis to clarify the clinicopathological

significance of SASPase mutation.

Physiological role of SASPase

Figure 8 summarizes our findings. In normal epidermis, non-

phosphorylated profilaggrin is orderly processed into filaggrin

and bundle keratin filaments at the ‘lower SC’, then degraded

into free amino acids which constitute most of the NMFs in the

‘upper SC’. SASPase deficiency causes incomplete linker

cleavage of profilaggrin resulting in an accumulation of trimeric

and dimeric profilaggrins slightly degraded from either N- or

C-terminal ends in the ‘lower SC’. Such aberrant profilaggrin

may bind to keratin filaments, finally degrade, and produce a

normal composition of free amino acids in the ‘upper SC’.

Finally, the SC of SASP�/� epidermis has an increased number

of layers and produces a wrinkled, dry, rough skin.

DISCUSSION

In our previous study, we reported increased fine wrinkles at the

side of the body in SASP�/� mice raised on a C57BL/6J

background. However, we were not able to distinguish any

morphological changes between SASPþ/� and SASP�/� mice,

possibly due to the thin epidermis of the back skin (Matsui et al,

2006). In this paper, we generated SASP�/� mice with Hos:HR-1

background. The increased thickness of the epidermis of these

mice enabled us to distinguish a clear difference in SC structure

and profilaggrin processing pattern in the SC. SASP�/� hairless

mice showed amarked decrease in SC hydration and an increased

number of electron dense layers in the SC. Such aberrant layers in

the SC consisted of an upper SC without filaggrin staining and a

lower SC stained by anti-filaggrin Ab with the accumulation of

aberrant dimeric and trimeric filaggrin, i.e. premature profilag-

grin processing (Figs 5B and 8). Interestingly, the composition


Figure 8. Schematic representation of the possible profilaggrin processing pathway observed in the SASPR/R and SASPS/S epidermis. In the SASPþ/þ

epidermis, highly phosphorylated profilaggrin expressed in the SG is dephosphorylated in the upper most SG. The linker sequence that connects each filaggrin

monomer is then cleaved by SASPase at the SG-to-SC transition via production of two kinds of intermediates (2DI and 3DI). Monomeric filaggrin strongly binds to

keratin filaments in the lower SC to form bundled keratin filaments. After citrullination, filaggrin is released from keratin and further degraded to form free amino

acids, which constitute most of the NMFs in the upper SC. In the SASP�/� epidermis, trimeric and dimeric profilaggrins slightly degraded from either N- or

C-terminal ends, accumulate because of incomplete linker sequence cleavage. Aberrantly processed profilaggrin binds to keratin filaments and degrades into free

amino acids without the production of monomeric filaggrin. Finally, aberrant SC causes wrinkled, dry, rough skin with decreased SC hydration. SC, stratum

corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.


and quantity of major free amino acids were not altered in the

dry skin-like SC of SASP�/� hairless mice (Fig 6).

Hydration of the SC plays an important role in maintaining

metabolic activity, enzyme activity, mechanical properties,

appearance and barrier function of the skin and is dependent on

(i) organization of the tight and semi-permeable barrier of

intercellular lamellar lipids, (ii) the diffusion path length created

by the SC layers and corneocyte envelopes, and (iii) the presence

of NMFs (Rawlings & Harding, 2004; Rawlings & Matts, 2005).

NMFs comprise up to 10% of the SC and are reported to be an

important natural humectants of the SC because of their

hygroscopic features (Rawlings & Harding, 2004; Rawlings &

Matts, 2005). Most NMFs are composed of free amino acids

derived from the degradation products of filaggrin and its

derivatives, PCA and urocanic acid (UCA). There are also non-

amino-acid-derived (non-filaggrin-derived) NMFs, such as

sugars, hyaluronic acid, urea, citrate, lactate, and glycerol

(Fluhr et al, 2008; Rawlings & Harding, 2004; Rawlings & Matts,

2005). Although we did not analyse the PCA or UCA of SASP�/�

hairless mice, there were no changes in free amino acids, which

suggests that filaggrin-derived NMFs were not altered. The

accumulation of aberrantly processed profilaggrin without

monomer filaggrin in the ‘lower SC’ accompanied by a normal

free amino acid content in the ‘upper SC’ in SASP�/� SC implies

disruption of several mechanisms for maintenance of SC

hydration other than the contribution of NMFs.

As described in the Introduction section, filaggrin is thought

to have two major functions: the formation of keratin

microfibrils in the lower SC and the production of NMFs in

the upper SC (Rawlings & Harding, 2004; Fig 8). It is possible

that dry skin of SASP�/� hairless mice is derived from the lower

SC where aberrantly processed filaggrin accumulates, but not

from the upper SC where the end-products of filaggrin (NMFs)

are located. In the normal epidermis of humans, rats and mice,

intermediate processing products of mouse filaggrin, 2DI and

3DI, have been reported, and they possess keratin binding

activity similar to monomeric filaggrin (Harding & Scott, 1983).

Thus, it is suggested that aberrant dimeric and trimeric filaggrins

in the SASP�/� SC also possess keratin binding activity. It is

widely believed that the SC hydration and physiological and

physical mechanics are closely linked and depend on keratin

structural organization. Accumulation of premature processed

profilaggrin, even if there is keratin binding activity, may alter

the cubic-like, rod-packing symmetry of keratin filaments at the

SG-to-SC transition and/or at the lower SC, and this may cause

alteration of the SC hydration level in the SASP�/� epidermis

(Norlen & Al-Amoudi, 2004). The SC of flaky tail mice, in which

filaggrin is absent in the cornified cell layers of the epidermis,

did not show decreased SC hydration nor an increased number

of layers in the SC (Fallon et al, 2009; Presland et al, 2000;

Scharschmidt et al, 2009). This suggests that aberrantly

processed profilaggrin and a marked decrease of mature

filaggrin affect the texture and hydration of the SC. The

importance of the amount of mature filaggrin for SC hydration

was reported by Ginger et al, who found an inverse relationship

between the profilaggrin-12-repeat allele and the occurrence of

self-perceived dry skin (Ginger et al, 2005).

SASPase may cleave other as yet unidentified substrates, in

addition to profilaggrin, resulting in decreased SC hydration via

abnormalities in the intercellular lamellar lipids barrier, the

diffusion path length and/or the composition of non-filaggrin-

derived NMFs. These possibilities can be examined by crossing

SASP�/� mice with filaggrin-deficient mice to examine whether

dry skin is derived from aberrant profilaggrin processing.

In normal, healthy controls and AD patients, we identified

several missense mutations, which may have affected the activity

of SASPase. Among them, we identified a loss-of-function

mutation of SASPase (V243A) in non-AD controls. This mutation

was located inside the protease domain and no longer showed any

activity at pH 6.0. Another missense mutation, V187I, was the

most frequently identified in the AD patients (three AD patients).

It was located outside of the protease domain at the autoproces-

sing site, the ‘P4’ position of the substrate recognition site. A

change of the amino acid at P4 resulted in decreased autoproces-

sing activity in vitro. GST-hSASP28(V187I) did not show any

autoprocessing activity in E. coli (neutral pH), suggesting that this

mutation had no activity in the cytoplasm of the SG, resulting in

the same loss-of-function effect as SASPase(V243A). SASPase, as a

retroviral aspartic protease, must undergo homodimeric forma-

tion for its protease activity (Bernard et al, 2005; Matsui et al,

2006). In the HIV protease, a subunit exchange reaction with a

catalytically defective protease results in 50% inhibition of

enzymatic activity (Darke, 1994). In the case of a heterozygous

loss-of-function mutation of bi-allelic expression, half of the

molecules are catalytically inactive and induce ‘heterodimeric

inhibition’ of SASPase activity, resulting in a quarter decrease of

total activity. The newly found, rare mutations of SASPase

(V187I)/(V243A) possibly behave in a dominant negativemanner

in the person who has the heterozygous mutation.

Of note, the SASP�/� mice showed decreased SC hydration

without alteration of TEWL, suggesting a decreased ability of

water retention in the SC under normal barrier function.

Although the TEWL is an important hallmark for skin barrier

function, TEWL is not necessarily correlated with dry skin

(Berry et al, 1999; Engelke et al, 1997; Wilhelm et al, 1991).

Decreased SC hydration is found in a number of diseases, such

as AD, eczema or psoriasis (Harding et al, 2000). There are a

number of human epidermal diseases that include the aberrant

expression and processing of profilaggrin to filaggrin (reviewed

in Dale et al, 1990). Therefore, it is possible that patients who do

not have a nonsense mutation of filaggrin, but who exhibit

xerosis or AD, might have an aberrant profilaggrin processing

pattern. Involvement of SASPase in progression of these

diseases from the aspect of the profilaggrin processing pathway

should be examined. The profilaggrin degradation pattern could

be useful for the diagnosis of xerosis and the early onset of AD.

Collectively, these results indicate that activity of SASPase

plays a key role in determining the texture of the SC by

modulating SC hydration as well as profilaggrin-to-filaggrin

processing. Moreover, these results, in combination with

clinicopathological investigations of epidermal diseases derived

from the aberrant processing of profilaggrin by SASPase

mutation, will provide a novel concept to dissect the complex

mechanisms of percutaneous antigen priming in atopic diseases.



MATERIALS AND METHODS

Reagents

Oligonucleotide primers were purchased from Sigma–Aldrich Japan

(Kyoto, Japan). N-terminal amino acid sequence analysis was

performed by Shimadzu Techno Research (Kyoto, Japan).

Antibodies

For immunofluorescence and immunoblotting, antibodies against

keratin 14, keratin 1, involucrin, loricrin and filaggrin were used

(Covance, Berkeley, CA). A polyclonal antibody against SASPase, anti-

SASP-C, which recognizes the C-terminus of both 28 kDa (human) and

32 kDa (mouse) SASPase, and anti-SASP-PR1 polyclonal antibody,

which recognizes both 14 kDa (human) and 15 kDa (mouse) SASPase,

have been described previously (Matsui et al, 2006).

Animals

Hairless mice (Hos/HR-1) were obtained from Hoshino Experimental

Animal Supply (Ibaragi, Japan). SASPþ/� mice of a C57BL/6J back-

ground were bred from six generations to a pure Hos/HR-1

background through the speed congenic services of the Central

Institute for Experimental Animals (Kawasaki, Japan). Mice hetero-

zygous for a SASPase deletion were interbred, and SASPþ/þ, SASPþ/�

and SASP�/� littermates were used for experiments. Wild-type (WT,

SASPþ/þ), SASPase heterogenic (SASPþ/�) and SASPase knockout

(SASP�/�) mice on a Hos/HR-1 backgrounds were used in the study.

All mice were maintained under specific pathogen-free (SPF)

conditions that are required for maintaining mouse colonies. All

animal procedures were approved by the Animal Studies Subcommit-

tee (Institutional Animal Care and Use Committee) of the Tokyo

Medical and Dental University and performed in accordance with their

guidelines. Basal SC hydration was measured with ASA-M1 (Asahi

Biomed, Tokyo, Japan) on the back skin of SASPþ/þ (n¼7) and SASP�/�

(n¼11) mice. From the same mice, TEWL measurements were taken

under basal conditions with a VAVO SCAN (Asahi Biomed). Experiments

were performed with adult female mice only (2–5 months old).

Tape-stripped epidermal extract

The dorsal and back skin of SASPþ/þ, SASPþ/� and SASP�/� hairless

mice were sequentially stripped with Scotch Book Tape 10 times (3M,

St. Paul, MN). Corneocytes adherent to the tape surface were eluted by

5ml of urea-buffer and concentrated by an Amicon-ultra 10 kDa

(Millipore) into 50ml. Protein concentrations were estimated by the

Bradford method. Samples were mixed with 25ml of 3� SDS sample

buffer.

Free amino acid analysis

Surface samples of SC were obtained by adhesive tape striping (Scotch

Book Tape) performed five times. Each tape corresponded to a skin

area of about 3�10 cm2 derived from anesthetized SASPþ/þ (n¼7)

and SASP�/� (n¼11) hairless mice. Water-soluble amino acids on the

adhesive tapes were extracted with 5ml 0.1% Triton X-100. After

sonication for 30min at 37 8C, amino acids were quantified using an

amino acid analyser (Hitachi model L-8500).

Autoprocessing assay

Fifty microlitres of GST-hSASP28 mutants (1.4mg/ml or 0.9mg/ml) in

buffer D (50mM phosphate buffer, pH 6.0, 0.7M NaCl) containing

1mM EDTA and protease inhibitor cocktail) was incubated at 378C.

Five-microlitres aliquots were recovered at different times during the


The paper explained

PROBLEM:

The SC is the outermost layer of the skin in terrestrial animals and

thus acts as a barrier against the external environment. It is

hydrated by endogenous substances to avoid desiccation;

however, the mechanisms responsible for maintaining hydration

of the SC remain unclear at the molecular level. Dry skin is a

common phenotype in patients with atopic dermatitis (AD).

Recent reports have indicated that the protein filaggrin is

mutated in ichthyosis vulgaris patients and is a major

predisposing factor for development of atopic eczema, asthma,

and allergies. Approximately 50 and 80% of European and

Japanese AD patients, respectively, have normal filaggrin alleles,

suggesting the presence of previously unidentified predisposing

factors.

RESULTS:

We produced ‘hairless’ mice that were deficient in the enzyme

skin-specific retroviral-like aspartic protease (SASPase). The

decreased activity of this enzyme in the mice resulted in dry skin

with an accumulation of incorrectly processed profilaggrin, a

precursor of the filaggrin protein. This incorrectly processed and

accumulated profilaggrin subsequently results in a marked

decrease of filaggrin production. We also demonstrated that

SASPase directly cleaved a profilaggrin linker peptide in vitro.

Several missense mutations were detected in 5 of 196 AD

patients and 2 of 28 normal individuals. Among these, the V243A

mutation resulted in complete ablation of protease activity

in vitro, while the V187I mutation induced a marked decrease in

SASPase activity.

IMPACT:

This is the first report demonstrating that a deficiency of the

protease SASPase is a likely cause of dry skin in vivo. We clarified

that the activity of SASPase plays a key role in determining the

texture of the SC by modulating SC hydration. This molecular

mechanism will provide clues in revealing the role of the SC in

terrestrial animals and how they adapted to life on land. Our

results also provide novel concepts to assist in determining the

complex pathophysiology of atopic dry skin.


incubation step, and the reaction was stopped by the addition of 20ml

Laemmli buffer. All the aliquots (5ml) were analysed by SDS-PAGE on

15% acrylamide gels followed by staining with CBB R-250. Semi-

quantification of hSASP14 was analysed densitometrically using

Adobe PhotoshopTM CS3.

Purification of recombinant hSASP14

All procedures were performed at 48C. Purified GST-hSASP28 was

dialysed against buffer D using NAP-10 (GE Healthcare, Japan) and

concentrated and frozen at �80 8C. Next, 300ml of concentrated

GST-SASP28 (5mg/ml) was incubated for 60min at 378C to

commence autoprocessing. Each sample was diluted in 1ml of buffer

D and passed over 200ml of Glutathione Sepharose 4B beads (GE

Healthcare) twice. The flow-through fraction containing hSASP14 was

collected and subjected to the protease assay.

Human filaggrin cleavage assay

Purified hSASP14 (419 pmol) was incubated with 3.6mM MBP-

hFilaggrin in 100ml of buffer D in the presence of 1mM EDTA and

protease inhibitor cocktail for 60min at 378C. After incubation, the

reactions were stopped by adding 50ml of 3� SDS sample buffer, and

10ml was subjected to SDS-PAGE. Cleaved fragments were subjected

to N-terminal amino acid sequencing.

Author contributionsTM conceived of the study, participated in its design and

coordination, carried out the analysis of knockout mice and the

biochemical studies and drafted the manuscript; KM and JK

carried out the mutation search; AK participated in the design of

the study and helped to draft the manuscript; HK and TE

conducted the human study; KH and ST helped to analyse the

knockout mice; SI carried out the electron microscopic analysis;

II and JI participated in the design and coordination of the study

and helped to draft the manuscript; MA conceived of the study,

participated in its design and coordination and helped to draft

the manuscript. All authors read and approved the final

manuscript.

AcknowledgementsWe thank Sayaka Katahira-Tayama and Itsumi Ohmori for

technical assistance. We thank Dr. Tsuyohi Hata (KOSE

Corporation) for helpful discussions. We thank KAN Research

Institute Inc. for providing materials. This work was

supported by a Grant-in-Aid for Scientific Research to TM

to AK, and to HK, ‘Program for Improvement of Research

Environment for Young Researchers’ from the Ministry of

Education, Culture, Sports, Science and Technology (MEXT)

of Japan to TM and to AK, research grants from the

Nakatomi Foundation, the Cosmetology Research Foundation

and the Naito Foundation to TM, the Keio University

Global Center of Excellence Program for In vivo Human

Metabolomic Systems Biology from MEXT and Health and

Labour Sciences Research Grants for Research on Allergic

Diseases and Immunology from the Ministry of Health,

Labour and Welfare.

Supporting information is available at EMBO Molecular

Medicine online.

The authors declare that they have no conflicts of interest.

For more information

OMIM: SKIN ASPARTIC PROTEASE:

http://www.ncbi.nlm.nih.gov/omim/611765

GeneCards:

http://www.genecards.org/cgi-bin/carddisp.pl?gene=ASPRV1

ReferencesBarker JN, Palmer CN, Zhao Y, Liao H, Hull PR, Lee SP, Allen MH, Meggitt SJ,

Reynolds NJ, Trembath RC et al (2007) Null mutations in the filaggrin gene

(FLG) determine major susceptibility to early-onset atopic dermatitis that

persists into adulthood. J Invest Dermatol 127: 564-567

Barnes KC (2010) An update on the genetics of atopic dermatitis:

scratching the surface in 2009. J Allergy Clin Immunol 125: 16-29 e11–11;

quiz 30-11

Bernard D, Mehul B, Thomas-Collignon A, Delattre C, Donovan M, Schmidt R

(2005) Identification and characterization of a novel retroviral-like aspartic

protease specifically expressed in human epidermis. J Invest Dermatol 125:

278-287

Berry N, Charmeil C, Goujon C, Silvy A, Girard P, Corcuff P, Montastier C (1999)

A clinical, biometrological and ultrastructural study of xerotic skin. Int J

Cosmet Sci 21: 241-252

Candi E, Tarcsa E, Digiovanna JJ, Compton JG, Elias PM, Marekov LN, Steinert

PM (1998) A highly conserved lysine residue on the head domain of type II

keratins is essential for the attachment of keratin intermediate filaments to

the cornified cell envelope through isopeptide crosslinking by

transglutaminases. Proc Natl Acad Sci USA 95: 2067-2072

Candi E, Schmidt R, Melino G (2005) The cornified envelope: a model of cell

death in the skin. Nat Rev Mol Cell Biol 6: 328-340

Dale BA, Holbrook KA, Steinert PM (1978) Assembly of stratum corneum basic

protein and keratin filaments in macrofibrils. Nature 276: 729-731

Dale BA, Resing KA, Lonsdale-Eccles JD (1985) Filaggrin: a keratin filament

associated protein. Ann NY Acad Sci 455: 330-342

Dale BA, Resing KA, Haydock PV (1990) Filaggrins. In: Cellular and Molecular

Biology of Intermediate Filaments, Goldman RD and Steinert PM, (eds), New

York and London, Plenum Press: pp 393-412.

Darke PL (1994) Stability of dimeric retroviral proteases. Methods Enzymol

241: 104-127

Denecker G, Hoste E, Gilbert B, Hochepied T, Ovaere P, Lippens S, Van den

Broecke C, Van Damme P, D’Herde K, Hachem JP et al (2007) Caspase-14

protects against epidermal UVB photodamage and water loss. Nat Cell Biol

9: 666-674

Elias PM, Steinhoff M (2008) ‘‘Outside-to-inside’’ (and now back to ‘‘outside’’)

pathogenic mechanisms in atopic dermatitis. J Invest Dermatol 128: 1067-

1070

Engelke M, Jensen JM, Ekanayake-Mudiyanselage S, Proksch E (1997) Effects

of xerosis and ageing on epidermal proliferation and differentiation. Br J

Dermatol 137: 219-225

Fallon PG, Sasaki T, Sandilands A, Campbell LE, Saunders SP, Mangan NE,

Callanan JJ, Kawasaki H, Shiohama A, Kubo A et al (2009) A homozygous

frameshift mutation in the mouse Flg gene facilitates enhanced

percutaneous allergen priming. Nat Genet 41: 602-608

Fluhr JW, Darlenski R, Surber C (2008) Glycerol and the skin: holistic approach

to its origin and functions. Br J Dermatol 159: 23-34

Ginger RS, Blachford S, Rowland J, Rowson M, Harding CR (2005) Filaggrin

repeat number polymorphism is associated with a dry skin phenotype. Arch

Dermatol Res 297: 235-241



Harding CR, Scott IR (1983) Histidine-rich proteins (filaggrins): structural and

functional heterogeneity during epidermal differentiation. J Mol Biol 170:

651-673

Harding CR, Watkinson A, Rawlings AV, Scott IR (2000) Dry skin,

moisturization and corneodesmolysis. Int J Cosmet Sci 22: 21-52

Hildenbrand M, Rhiemeier V, Hartenstein B, Lahrmann B, Grabe N, Angel P,

Hess J (2010) Impaired skin regeneration and remodeling after cutaneous

injury and chemically induced hyperplasia in taps-transgenic mice. J Invest

Dermatol 130: 1922-1930

Irvine AD (2007) Fleshing out filaggrin phenotypes. J Invest Dermatol 127:

504-507

Ishida-Yamamoto A, Senshu T, Eady RA, Takahashi H, Shimizu H, Akiyama M,

Iizuka H (2002) Sequential reorganization of cornified cell keratin filaments

involving filaggrin-mediated compaction and keratin 1 deimination. J

Invest Dermatol 118: 282-287

Kamata Y, Taniguchi A, YamamotoM, Nomura J, Ishihara K, Takahara H, Hibino

T, Takeda A (2009) Neutral cysteine protease bleomycin hydrolase is

essential for the breakdown of deiminated filaggrin into amino acids. J Biol

Chem 284: 12829-12836

Leyvraz C, Charles RP, Rubera I, Guitard M, Rotman S, Breiden B, Sandhoff K,

Hummler E (2005) The epidermal barrier function is dependent on the

serine protease CAP1/Prss8. J Cell Biol 170: 487-496

List K, Szabo R, Wertz PW, Segre J, Haudenschild CC, Kim SY, Bugge TH (2003)

Loss of proteolytically processed filaggrin caused by epidermal deletion of

Matriptase/MT-SP1. J Cell Biol 163: 901-910

Matsui T, Hayashi-Kisumi F, Kinoshita Y, Katahira S, Morita K, Miyachi Y, Ono Y,

Imai T, Tanigawa Y, Komiya T et al (2004) Identification of novel

keratinocyte-secreted peptides dermokine-alpha/-beta and a new stratified

epithelium-secreted protein gene complex on human chromosome

19q13.1. Genomics 84: 384-397

Matsui T, Kinoshita-Ida Y, Hayashi-Kisumi F, Hata M, Matsubara K, Chiba M,

Katahira-Tayama S, Morita K, Miyachi Y, Tsukita S (2006) Mouse homologue

of skin-specific retroviral-like aspartic protease involved in wrinkle

formation. J Biol Chem 281: 27512-27525

McGrath JA, Uitto J (2008) The filaggrin story: novel insights into skin-barrier

function and disease. Trends Mol Med 14: 20-27

Mechin MC, Enji M, Nachat R, Chavanas S, Charveron M, Ishida-Yamamoto A,

Serre G, Takahara H, Simon M (2005) The peptidylarginine deiminases

expressed in human epidermis differ in their substrate specificities and

subcellular locations. Cell Mol Life Sci 62: 1984-1995

Nachat R, MechinMC, Takahara H, Chavanas S, CharveronM, Serre G, SimonM

(2005) Peptidylarginine deiminase isoforms 1–3 are expressed in the

epidermis and involved in the deimination of K1 and filaggrin. J Invest

Dermatol 124: 384-393

Nomura T, Akiyama M, Sandilands A, Nemoto-Hasebe I, Sakai K, Nagasaki A,

Ota M, Hata H, Evans AT, Palmer CN et al (2008) Specific filaggrin mutations

cause ichthyosis vulgaris and are significantly associated with atopic

dermatitis in Japan. J Invest Dermatol 128: 1436-1441

Norlen L, Al-Amoudi A (2004) Stratum corneum keratin structure, function,

and formation: the cubic rod-packing and membrane templating model. J

Invest Dermatol 123: 715-732

Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, Goudie DR,

Sandilands A, Campbell LE, Smith FJ et al (2006) Common loss-of-function

variants of the epidermal barrier protein filaggrin are a major predisposing

factor for atopic dermatitis. Nat Genet 38: 441-446

Pearton DJ, Nirunsuksiri W, Rehemtulla A, Lewis SP, Presland RB, Dale BA

(2001) Proprotein convertase expression and localization in epidermis:

evidence for multiple roles and substrates. Exp Dermatol 10: 193-203

Presland RB, Boggess D, Lewis SP, Hull C, Fleckman P, Sundberg JP (2000) Loss

of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model

for the filaggrin-deficient skin disease ichthyosis vulgaris. J Invest Dermatol

115: 1072-1081

Presland RB, Rothnagel JA, Lawrence OT (2006) Profilaggrin and the fused

S100 family of calcium-binding proteins. In: In Skin Barrier, Elias PM and

Feingold KR, (eds) New York, Taylor and Francis: pp 111-140.

Rawlings AV, Harding CR (2004) Moisturization and skin barrier function.

Dermatol Ther 17: 43-48

Rawlings AV, Matts PJ (2005) Stratum corneum moisturization at the

molecular level: an update in relation to the dry skin cycle. J Invest Dermatol

124: 1099-1110

Resing KA, Walsh KA, Haugen-Scofield J, Dale BA (1989) Identification of

proteolytic cleavage sites in the conversion of profilaggrin to filaggrin in

mammalian epidermis. J Biol Chem 264: 1837-1845

Resing KA, al-Alawi N, Blomquist C, Fleckman P, Dale BA (1993) Independent

regulation of two cytoplasmic processing stages of the intermediate

filament-associated protein filaggrin and role of Ca2þ in the second stage. J

Biol Chem 268: 25139-25145

Resing KA, Johnson RS, Walsh KA (1993) Characterization of protease

processing sites during conversion of rat profilaggrin to filaggrin.

Biochemistry 32: 10036-10045

Resing KA, Thulin C, Whiting K, al-Alawi N, Mostad S (1995) Characterization

of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase

of epidermis. J Biol Chem 270: 28193-28198

Rhiemeier V, Breitenbach U, Richter KH, Gebhardt C, Vogt I, Hartenstein B,

Furstenberger G, Mauch C, Hess J, Angel P (2006) A novel aspartic

proteinase-like gene expressed in stratified epithelia and squamous cell

carcinoma of the skin. Am J Pathol 168: 1354-1364

Sandilands A, Terron-Kwiatkowski A, Hull PR, O’Regan GM, Clayton TH,Watson

RM, Carrick T, Evans AT, Liao H, Zhao Y et al (2007) Comprehensive analysis

of the gene encoding filaggrin uncovers prevalent and rare mutations in

ichthyosis vulgaris and atopic eczema. Nat Genet 39: 650-654

Sandilands A, Sutherland C, Irvine AD, McLean WH (2009) Filaggrin in the

frontline: role in skin barrier function and disease. J Cell Sci 122: 1285-1294

Sasaki T, Kudoh J, Ebihara T, Shiohama A, Asakawa S, Shimizu A, Takayanagi A,

Dekio I, Sadahira C, Amagai M et al (2008) Sequence analysis of filaggrin

gene by novel shotgunmethod in Japanese atopic dermatitis. J Dermatol Sci

51: 113-120

Scharschmidt TC, ManMQ, Hatano Y, Crumrine D, Gunathilake R, Sundberg JP,

Silva KA, Mauro TM, HupeM, Cho S et al (2009) Filaggrin deficiency confers a

paracellular barrier abnormality that reduces inflammatory thresholds to

irritants and haptens. J Allergy Clin Immunol 124: 496-506, 506 e491-496

Smith FJ, Irvine AD, Terron-Kwiatkowski A, Sandilands A, Campbell LE, Zhao Y,

Liao H, Evans AT, Goudie DR, Lewis-Jones S et al (2006) Loss-of-function

mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat

Genet 38: 337-342

Steinert PM, Marekov LN (1995) The proteins elafin, filaggrin, keratin

intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are

isodipeptide cross-linked components of the human epidermal cornified

cell envelope. J Biol Chem 270: 17702-17711

Tarcsa E, Marekov LN, Mei G, Melino G, Lee SC, Steinert PM (1996) Protein

unfolding by peptidylarginine deiminase. Substrate specificity and

structural relationships of the natural substrates trichohyalin and filaggrin.

J Biol Chem 271: 30709-30716

Thulin CD, Walsh KA (1995) Identification of the amino terminus of human

filaggrin using differential LC/MS techniques: implications for profilaggrin

processing. Biochemistry 34: 8687-8692

Thulin CD, Taylor JA, Walsh KA (1996) Microheterogeneity of human filaggrin:

analysis of a complex peptide mixture using mass spectrometry. Protein Sci

5: 1157-1164

Wakeland E, Morel L, Achey K, Yui M, Longmate J (1997) Speed congenics: a

classic technique in the fast lane (relatively speaking). Immunol Today 18:

472-477

Watt FM (1989) Terminal differentiation of epidermal keratinocytes. Curr

Opin Cell Biol 1: 1107-1115

Wilhelm KP, Cua AB, Maibach HI (1991) Skin aging. Effect on transepidermal

water loss, stratum corneum hydration, skin surface pH, and casual sebum

content. Arch Dermatol 127: 1806-1809

Yamazaki M, Ishidoh K, Suga Y, Saido TC, Kawashima S, Suzuki K, Kominami E,

Ogawa H (1997) Cytoplasmic processing of human profilaggrin by active

mu-calpain. Biochem Biophys Res Commun 235: 652-656



saspase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing

Documents